Treatment of cancers with acquired resistance to kit inhibitors

ABSTRACT

The present invention provides compositions and methods for treating cancers which have acquired resistance to a KIT inhibitor by administering effective amounts of DAST.

DESCRIPTION OF THE INVENTION

Cancer is a class of diseases characterized by two heritable properties:(1) uncontrolled cell division and (2) the ability of these cells toinvade other tissues, either by direct growth into adjacent tissue(invasion) or by migration of cells to distant sites (metastasis). Thehyper-proliferative properties initially give rise to a tumor orneoplasm. A tumor is considered a cancer when its cells acquire theability to invade surrounding tissues, e.g., by breaking loose andentering the blood or lymph systems, or by forming secondary tumors atother sites in the body. The unregulated growth is caused by damagedDNA, resulting in mutations to vital genes that control cell division,the cell cycle, among other functions. One or more of these mutations,which can be inherited or acquired, can lead to uncontrolled celldivision and cancer.

Cancers can be classified according to the tissue and cell type fromwhich they arise. Cancers developing from epithelial cells are calledcarcinomas, and those from connective and muscle cells are calledsarcomas. Additional cancers include those arising from hematopoieticcells (e.g., leukemia) and cancers of the nervous system.

In general, cancers appear to arise during a process in which an initialpopulation of abnormal cells evolve into more aberrant cells throughsuccessive cycles of mutation and selection. More than 100 differentgenes have been identified which, when mutant, result in cancer. Theseso-called cancer-critical genes fall into two broad classes: oncogenesand tumor suppressor genes. Many cancer-critical genes play a role inthe regulation of cell divisions, a highly complicated process involvingmultiple and parallel pathways. These include growth factors, cytokines,hormones, etc.

Cancer can cause many different symptoms, depending on the site andcharacter of the malignancy and whether there is metastasis. Adefinitive diagnosis usually requires the microscopic examination oftissue obtained by biopsy. Once diagnosed, cancer is usually treatedwith surgery, chemotherapy and/or radiation.

If untreated, most cancers eventually cause death. Cancer is one of theleading causes of death in developed countries. It is estimated by theNational Cancer Institute that approximately 9.8 million Americans werealive in January 2001 with a history of cancer. About 1,372,910 newcases of cancer were expected to be diagnosed in 2005, alone. In 2005,almost 600,000 Americans died of cancer, about 1 out of every 4 deaths.Many forms of cancer are associated with environmental factors, whichmay be avoidable. Smoking tobacco leads to more cancers than any otherenvironmental factor.

Kinase inhibitors are being used successfully to treat cancers (e.g.,Drevs et al., Current Drug Targets, 2003, 4, 113-121). However, somepatients acquire a resistance to the drug's activity. In one embodiment,the present invention provides methods of treating a cancer in a subjectin need thereof, comprising administering an effective amount of DAST toa subject having a cancer, wherein said cancer has acquired resistanceto a KIT tyrosine kinase inhibitor. A tyrosine kinase inhibitor is adrug (i.e., a chemical compound) that blocks or reduces its kinaseactivity. Generally, a “tyrosine kinase activity” refers to the abilityof the tyrosine kinase to auto-phosphorylate itself ortrans-phosphorylate receptor subunits (or other substrates) bycatalyzing the transfer of a phosphate from ATP (or another phosphatedonor) to a tyrosine residue.

There are a number of well-documented instances where cancers haveacquired resistance to a kinase inhibitor which previously hadsuccessfully been used to treat the cancer. The term “acquiredresistance” indicates that the cancer becomes resistant and/orsubstantially less response to the effects of the drug after beingexposed to it for a certain period of time. For example,gastrointestinal stromal tumors (GIST), a mesenchymal tumor of theintestinal tract, and chronic myelogenous leukemia (CML) are treatedwith imatinib (STI571 or Gleevac), a tyrosine kinase inhibitor thatinhibits the kinase activity of BCR-ABL, ABL, KIT, and PDGFR. It hasshown been shown that, while patients may benefit from the treatmentinitially, many patients subsequently develop resistance to the agent.In some cases, this acquired resistance has been shown to result from asecondary mutation in the gene associated with the cancer. For example,many GIST patients have an activating mutation in either the KIT orPDGFRA gene. A study of GIST patients with acquired resistance toimatinib showed secondary mutations in the KIT kinase domain. See, e.g.,Antonescu et al, Clin. Cancer Res., 11(11):41824190, 2005 and Heinrichet al., J. Clin. Oncology, 24(29), 4764-4774, 2006 A second sitemutation in BCR-ABL is the predominant mechanism of imiatinib resistancein CML. See, e.g., Gorre et al., Science, 293:876-880, 2001. Acquiredresistance has also been observed with other cancer drugs, includingpatients treated with EGFR-kinase inhibitors, such as gefitinib (Iressa)or erlotinib (Tarceva). See, e.g., Kobayshi et al., N. Engl. J. Med.,352:786-792, 2005. Pao et al. (PLoS Med., 2, e73, 2005) observed thatpatients with progressing lung tumors contained, in addition to aprimary drug-sensitive mutation in EGFR, a secondary mutation in thekinase domain which led to drug-resistance.

Examples of KIT inhibitors to which drug resistance can be acquiredincludes, but is not limited to, e.g., imatinib mesylate, andderivatives and salts thereof; PP1(4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine);MLN518 (CT53518); PD180970; SU112481 SU5416; SU5414; SU6597; SU6663;SU6561. See, also, Krystal et al., Cancer Res., 2001, 61:3660-3668.

Resistance mutations often occur in the kinase catalytic domaininterfering or weakening the interaction with its inhibitor. Resistancesecondary mutations for KIT have been reported. These secondarymutations often occur in the “gatekeeper” residue, the amino acidresidue that “guards” the ATP-binding pocket and which also can comprisethe site which interacts with the inhibitor. See, e.g., Noble et al.,Science, 303: 1800-1805, 2004.

While not being bound to any mechanism, examples of mutations in the KITgene which are associated with resistance or acquired resistanceinclude, but are not limited to, e.g., mutations in Exons 13, 14, and or17; mutations at residues 654, 670, 716, 816, 820, 822, and 823,residues about 650-654, residues about 670-674, residues about 816-824,in the A-loop (activation), such as V654A (Exon 13), T670I (Exon 14),T670E, D716N, 5709F (Exon 14), D816G, D816E (Exon 17), C809G, D816H,D816V, D820A, D820E, D820Y, D820G N822K, Y823D (Exon 17), and/ordeletions and other amino acid substitutions at such positions, oradjacent positions. Generally, any cancer having a primary and/orsecondary KIT gene mutation associated with resistance or acquiredresistance to a KIT inhibitor can be treated with a compound inaccordance with the present invention.

As shown in the examples, mutations can decrease the affinity of akinase inhibitor, such as Imatinib (Gleevec), for the c-KIT protein,thereby decreasing the therapeutic efficacy of the drug. Table 1 showsspecific examples where the binding affinity of Imatinib (Gleevec)decreased. Any disorder in which the affected tissue (e.g., cancer)becomes resistant or less responsive to a KIT inhibitor can be treatedwith DAST or derivatives thereof.

KIT (also known as c-kit, mast cell growth factor receptor, or stem cellgrowth factor receptor) is the human homology of the provirus of theHardy-Zuckerman 4 feline sarcoma virus. KIT encodes a transmembranetyrosine kinase receptor which is expressed in a number of tissues, andis required for normal hematopoiesis, melanogenesis, and gametogenesis.The gene itself, is mapped to 4q11-q12, includes 21 exons, and isalternatively spliced. See, e.g., Vandenbark et al., Oncogene,7:1259-1266, 1992.

Over-expression and/or gain-of-function mutations in KIT can result inligand-independent tyrosine kinase activity, autophosphorylation of KIT,uncontrolled cell proliferation, and stimulation of downstream signalingpathways. For example, KIT was overexpressed in both malignant andbenign gastrointestinal stromal tumors (GIST) tumors. See, e.g., Koon etal., Gut, 2004, 53:235-240. KIT is also expressed in acute myeloidleukemia, mast cell tumors, SCLC, germ cell tumors, breast cancer, andneuroblastoma.

Activating mutations in the KIT gene are associated with many types ofGIST, the most common mesenchymal neoplasm in the human digestive tract.For example, Hirota et al., Science, 279:577-580, 1998, showed that of49 mesenchymal tumors, 94% of them expressed an activated KIT. GISTsinclude a spectrum of tumors, including both benign and malignant types,and which occur at all levels of the gastrointestinal tract (e.g.,stomach, small intestine, large intestine, rectum, etc.)

Cancers which are initially sensitive to a KIT inhibitor, but which haveacquired resistance to it, can be treated in accordance with the presentinvention. Cancers having mutations in Exon 11 (from amino acidpositions 550-582; see, e.g., Table 2) of the KIT gene are of particularrelevance, and more preferably within codons 550-560. This region canalso be referred to as the juxtamembrane domain. Specific examplesinclude, but are not limited to: 1) deletion of amino acid residues557-558; 2) deletion of amino acid residues 551-555; 3) deletion ofamino acid residues 550-558; 4) deletion of amino acid residues 559-560;5) deletion of amino acid residues 557-561; 6) deletion of amino acidresidues 554-558; 7) deletion of amino acid residues 552-557; 8)mutations at residue 559, including V559D, V559A, or V559G; 9) mutationsat residue 560, including V560D, V560E, or V560G; 10) W557S, alone, orin combination with a deletion of amino acids 552-556; 11) mutations atamino acid residue 557, including W557R; 12) mutations at amino acidresidue 576, including L576P; 13) InsQL576-577. These mutations can bealone, or combined with other mutations, including with any of thespecifically mentioned mutations. See, also, Lasota et al., Am. J.Path., 154:53-60, 1999.

Drug resistant cancers associated with other KIT mutations can betreated as well, especially those which are sensitive to KIT inhibitors.These include, e.g., systemic mastocytosis, e.g., having a F522Cmutation (Akin et al., Blood, 2004, 193:3222-3225) and K5091 (Zhang etal., 2005, Leuk. Res., September 21); testicular seminomas, e.g., havingimatinib mesylate sensitive mutations at amino acid residues 822 and823, such as N822K and Y823D (e.g., Kemmer et al., Am. J. Pathol., 2004,164:305-313, 2004).

Analysis of the gene mutations associated with cancer (e.g., GIST)having KIT mutation can be routinely determined. For example, PCR can beutilized to amplify specific regions using the published sequences ofthe human KIT gene. See, e.g., Andre et al., Genomics, 1997, 39:216-226.For amplification of Exon 11, see, e.g., Lasota et al., Am. J. Path.,154:53-60, 1999.

Diarylureas are a class of serine-threonine kinase inhibitors as well astyrosine kinase inhibitors known in the art. The following publicationsillustrate their utility as active ingredients in pharmaceuticalcompositions for the treatment of hyper-proliferative diseases, such ascancer:

-   Smith al., Bioorg. Med. Chem. Lett. 2001, 11, 2775-2778,-   Lowinger et al., Clin. Cancer Res. 2000, 6(suppl.), 335-   Lyons et al., Endocr.-Relat. Cancer 2001, 8, 219-225.-   Riedl et al., Book of Abstracts, 92^(nd) AACR Meeting, New Orleans,    La., USA, abstract 4956.-   Khire et al., Book of Abstracts, 93^(rd) AACR Meeting, San    Francisco, Calif., USA, abstract 4211.-   Lowinger et al., Curr. Pharm. Design 2002, 8, 99-110.-   Carter et al., Book of Abstracts, 92^(nd) AACR Meeting, New Orleans,    La., USA, abstract 4954.-   Vincent et al., Book of Abstracts, 38^(th) ASCO Meeting, Orlando,    Fla., USA, abstract 1900.-   Hilger et al., Book of Abstracts, 38^(th) ASCO Meeting, Orlando,    Fla., USA, abstract 1916.-   Moore et al., Book of Abstracts, 38^(th) ASCO Meeting, Orlando,    Fla., USA, abstract 1816.-   Strumberg et al., Book of Abstracts, 38^(th) ASCO Meeting, Orlando,    Fla., USA, abstract 121.

Omega-Carboxyaryl diphenyl ureas are disclosed in WO00/42012 (publishedJul. 20, 2000) and WO00/41698 (published Jul. 20, 2000). DAST, thediphenyl urea referred to as4{4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-fluorophenoxy}-pyridine-2-carboxylicacid methyl amide herein, is disclosed in WO05/009961 (published Feb. 3,2005) and is described as a potent inhibitor of raf, VEGFR-2, p38, andPDGFR kinases. These enzymes are all molecular targets of interest forthe treatment of hyper-proliferative diseases, including cancer. Soliddispersions of DAST are described in WO06/026500 (published Mar. 9,2006).

Nonetheless, the present invention relates to using DAST to treat acancer, such as those mentioned above, which have acquired resistance toa KIT inhibitor, irrespective of the molecular mechanism responsible forit.

The present invention provides methods of treating cancers comprising,e.g., comprising administering to a subject in need thereof an effectiveamount of DAST, wherein the cancer is treated.

Examples of cancers that can be treated with imatinib, include, but notlimited to: Accelerated Phase Chronic Myelogenous Leukemia; AcuteErythroid Leukemia; Acute Lymphoblastic Leukemia; Acute LymphoblasticLeukemia in Remission; Acute Lymphocytic Leukemia; Acute Monoblastic andAcute; Monocytic Leukemia; Acute Myelogenous Leukemia; Acute MyeloidLeukemia; Adenocarcinoma of the Prostate; Adenoid Cystic Carcinoma ofthe Head and Neck; Advanced Gastrointestinal Stromal Tumor; AgnogenicMyeloid; Metaplasia; Anaplastic Oligodendroglioma; Astrocytoma; B-CellAdult Acute Lymphoblastic Leukemia; Blastic Phase Chronic MyelogenousLeukemia; Bone Metastases; Brain Tumor; Breast Cancer; Cancer; CentralNervous System Cancer; Childhood Acute Lymphoblastic Leukemia; ChildhoodAcute Lymphoblastic Leukemia in Remission; Childhood Central NervousSystem Germ Cell Tumor; Childhood Chronic Myelogenous Leukemia;Childhood Soft Tissue Sarcoma; Chordoma; Chronic Eosinophilic Leukemia(CEL); Chronic Idiopathic Myelofibrosis; Chronic Myelogenous Leukemia;Chronic Myeloid Leukemia; Chronic Myelomonocytic Leukemia; Chronic PhaseChronic Myelogenous Leukemia; Colon Cancer; Colorectal Cancer;Dermatofibrosarcoma; Dermatofibrosarcoma Protuberans (DFSP); DesmoidTumor; Eosinophilia; Epidemic Kaposi's Sarcoma; EssentialThrombocythemia; Ewing's Family of Tumors; Extensive Stage Small CellLung Cancer; Fallopian Tube Cancer; Familiar Hypereosinophilia;Fibrosarcoma; Gastric Adenocarcinoma; Gastrointestinal Neoplasm;Gastrointestinal Stromal Tumor; Glioblastoma; Glioma; Gliosarcoma; GradeI Meningioma; Grade II Meningioma; Grade Ill Meningioma; Hematopoieticand Lymphoid Cancer; High-Grade Childhood Cerebral Astrocytoma;Hypereosinophilic Syndrome; Idiopathic Pulmonary Fibrosis; L1 AdultAcute Lymphoblastic Leukemia; L2 Adult Acute Lymphoblastic Leukemia;Leukemia, Lymphocytic, Acute L2; Leukemia, Myeloid, Chronic; Leukemia,Myeloid, Chronic Phase; Liver Dysfunction and Neoplasm; Lung Disease;Lymphoid Blastic Phase of Chronic Myeloid Leukemia; Male Breast Cancer;Malignant Fibrous Histiocytoma; Mastocytosis; MeningealHemangiopericytoma; Meningioma; Meningioma; Meningioma; MetastaticCancer; Metastatic Solid Tumors; Myelofibrosis; Myeloid Leukemia,Chronic; Myeloid Leukemia, Chronic Accelerated-Phase; Myeloid Leukemia,Chronic, Chronic-Phase; Myeloid Metaplasia; Myeloproliferative Disorder(MPD) with Eosinophilia; Neuroblastoma; Non-T, Non-B Childhood AcuteLymphoblastic Leukemia; Oligodendroglioma; Osteosarcoma; Ovarian GermCell Tumor; Ovarian Low Malignant Potential Tumor; Ovarian Neoplasms;Pancreatic Cancer; Pelvic Neoplasms; Peritoneal Cavity Cancer;Peritoneal Neoplasms; Philadelphia Chromosome Positive ChronicMyelogenous Leukemia; Philadelphia Positive Acute LymphoblasticLeukemia; Philadelphia Positive Chronic Myeloid Leukemia in MyeloidBlast Crisis; Polycythemia Vera; Pulmonary Fibrosis; Recurrent AdultBrain Tumor; Recurrent Adult Soft Tissue Sarcoma; Recurrent BreastCancer; Recurrent Colon Cancer; Recurrent Esophageal Cancer; RecurrentGastric Cancer; Recurrent Glioblastoma Multiforme (GBM); RecurrentKaposi's Sarcoma; Recurrent Melanoma; Recurrent Merkel Cell Carcinoma;Recurrent Ovarian Epithelial Cancer; Recurrent Pancreatic Cancer;Recurrent Prostate Cancer; Recurrent Rectal Cancer; Recurrent SalivaryGland Cancer; Recurrent Small Cell Lung Cancer; Recurrent Tumors of theEwing's Family; Recurrent Uterine Sarcoma; Relapsing Chronic MyelogenousLeukemia; Rheumatoid Arthritis; Salivary Gland Adenoid Cystic Carcinoma;Sarcoma; Small Cell Lung Cancer; Stage II Melanoma; Stage II Merkel CellCarcinoma; Stage III Adult Soft Tissue Sarcoma; Stage III EsophagealCancer; Stage III Merkel Cell Carcinoma; Stage III Ovarian EpithelialCancer; Stage III Pancreatic Cancer; Stage III Salivary Gland Cancer;Stage IIIB Breast Cancer; Stage IIIC Breast Cancer; Stage IV Adult SoftTissue Sarcoma; Stage IV Breast Cancer; Stage IV Colon Cancer; Stage IVEsophageal Cancer; Stage IV Gastric Cancer; Stage IV Melanoma; Stage IVOvarian Epithelial Cancer; Stage IV Prostate Cancer; Stage IV RectalCancer; Stage IV Salivary Gland Cancer; Stage IVA Pancreatic Cancer;Stage IVB Pancreatic Cancer; Systemic Mastocytosis; T-Cell ChildhoodAcute Lymphoblastic Leukemia; Testicular Cancer; Thyroid Cancer;Unresectable or Metastatic Malignant Gastrointestinal Stromal Tumor(GIST); Unspecified Adult Solid Tumor; Untreated Childhood Brain StemGlioma; Uterine Carcinosarcoma, and Uterine Sarcoma.

The phrase “effective amount” indicates the amount of DAST which iseffective to treat any symptom or aspect of the cancer. Effectiveamounts can be determined routinely. Further guidance on dosages andadministration regimens is provided below.

The term “treating” is used conventionally, e.g., the management or careof a subject for the purpose of combating, alleviating, reducing,relieving, improving, etc., one or more of the symptoms associated witha cancer, including all cancers mentioned herein. Administeringeffective amounts of DAST can treat one or more aspects of the cancerdisease, including, but not limited to, causing tumor regression;causing cell death; causing apoptosis; causing necrosis; inhibiting cellproliferation; inhibiting tumor growth; inhibiting tumor metastasis;inhibiting tumor migration; inhibiting tumor invasion; reducing diseaseprogression; stabilizing the disease; reducing or inhibitingangiogenesis; prolonging patient survival; enhancing patient's qualityof life; reducing adverse symptoms associated with cancer; and reducingthe frequency, severity, intensity, and/or duration of any of theaforementioned aspects.

Any cancer can be treated in accordance of the present invention,irrespective of the type or cause of the cancer, and irrespective of thegenetic lesions associated with. Examples of cancers which can betreated include, but are not limited to, GIST, acute myeloid leukemia,mast cell tumors, SCLC, germ cell tumors, breast cancer, neuroblastoma,sinonasal lymphoma, etc.

Cancers which can be treated include, e.g., cancers which are primary;which arise from a primary tumor at a secondary metastatic site; whichhave been treated by surgery (e.g., entirely removed, surgicalresection, etc); which have been treated by chemotherapy, radiation,radio frequency ablation, and/or any other adjunct to drug therapy. Anysubject can be in accordance with the present invention, including,e.g., mammals, such as mice, rats, dogs, cats, non-human primates,monkeys, and humans.

The ability of DAST to treat a cancer with acquired resistance to a KITinhibitor can be routinely determined. For example, the IL-3-dependentmurine hematopoietic cell line, Ba/F3, can be cultured independently ofIL-3 when transfected with constitutively active KIT (e.g., having adeletion of amino acid residues 557-558). See, e.g., Tsujimura et al.,Blood, 1999, 93:1319-1329. In the presence of a KIT inhibitor, such asimatinib, cells expressing the constitutively active KIT polypeptideundergo cell death as a result of KIT inhibition. The presence of asecond mutation that confers resistance to the KIT inhibitor rescues thecells. Cells expressing the double-mutation (activating; KIT resistance)are cultured in the presence of DAST. Those cells which die aresensitive to DAST, indicating its usefulness in treating patients whohave acquired resistance to the KIT inhibitor.

Specific examples of cancers which can be treated in accordance with thepresent invention include cancers have a deletion of residues 557-558,and which have at least one of the following mutations: V654A, T670I,D820Y, N822K, and Y823D.

The present invention also provides methods of determining whether totreat a subject having cancer with DAST, comprising determining thepresence of a mutation in a KIT gene, wherein said mutation is anactivating and/or KIT-inhibitor resistance mutation, and administeringDAST to a subject having one or more pre-determined mutations.Activating and KIT-inhibitor resistance mutations have been describedabove. A subject who is resistant to a KIT inhibitor can be screened forthe presence of an activating and/or resistance mutation (such as thoselisted above), a subject having the mutation(s) can be treated withDAST.

The term “DAST” as used herein refers to the compound:4{4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-fluorophenoxy}-pyridine-2-carboxylicacid methylamide of the formula I below including all polymorphs,hydrates, solvates, pharmaceutically acceptable salts or combinationsthereof. Also included are the metabolites of4{4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-fluorophenoxy}-pyridine-2-carboxylicacid methylamide and prodrugs of4{4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-fluorophenoxy}-pyridine-2-carboxylicacid methylamide prepared by conventional techniques.

Suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include salts of inorganic and organic acids,such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoricacid, methanesulphonic acid, trifluoromethanesulfonic acid,benzenesulfonic acid, 1-naphthalenesulfonic acid, 2-naphthalenesulfonicacid, acetic acid, trifluoroacetic acid, malic acid, tartaric acid,citric acid, lactic acid, oxalic acid, succinic acid, fumaric acid,maleic acid, benzoic acid, salicylic acid, phenylacetic acid, andmandelic acid. In addition, pharmaceutically acceptable salts includesalts of inorganic bases, such as salts containing alkaline cations(e.g., Li⁺ Na⁺or K⁺), alkaline earth cations (e.g., Mg⁺², Ca⁺² or Ba⁺²),the ammonium cation, as well as acid salts of organic bases, includingaliphatic and aromatic substituted ammonium, and quaternary ammoniumcations, such as those arising from protonation or peralkylation oftriethylamine, N, N-diethylamine, N, N-dicyclohexylamine, lysine,pyridine, N,N-dimethylaminopyridine (DMAP), 1,4-diazabiclo[2.2.2]octane(DABCO), 1,5-diazabicyclo[4.3.0]non-5-ene (DBN) and1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).

Solvates for the purposes of the invention are those forms of thecompound where solvent molecules form a complex in the solid state andinclude, but are not limited to for example ethanol and methanol.Hydrates are a specific form of solvates, where the solvent molecule iswater.

The metabolites of DAST include oxidized derivatives wherein one or moreof the urea nitrogens shown in of Formula I are substituted with ahydroxyl group. The metabolites of DAST also include analogs where themethylamide group shown in Formula I is hydroxylated then de-methylatedby metabolic degradation. The metabolites of DAST further includeoxidized derivatives where the pyridine nitrogen atom shown in ofFormula I is in the N-oxide form (e.g. carries a hydroxy substituent)leading to those structures referred to in the art as 1-oxo-pyridine and1-hydroxy-pyridine.

DAST can be further modified with labile functional groups that arecleaved after in vivo administration to furnish the parent active agentand the pharmacologically inactive derivatizing (functional) group.These derivatives, commonly referred to as prodrugs, can be used, forexample, to alter the physicochemical properties of the active agent, totarget the active agent to a specific tissue, to reduce undesirable sideeffects and/or to alter the pharmacokinetic and pharmacodynamicproperties of the active agent (e.g., solubility, absorption,biostability and release time. see “Pharmaceutical Dosage Form and DrugDelivery Systems” (Sixth Edition), edited by Ansel et al., published byWilliams & Wilkins, pages 27-29, (1995) which is hereby incorporated byreference),

Suitable including N-dealkylation, O-dealkylation, aliphatichydroxylation, aromatic hydroxylation, N-oxidation, S-oxidation,deamination, hydrolysis reactions, glucuronidation, sulfation andacetylation (see Goodman and Gilman's The Pharmacological Basis ofTherapeutics (Ninth Edition), editor Molinoff et al., pub. byMcGraw-Hill, pages 11-13, (1996), which is hereby incorporated byreference).

Suitable prodrugs of DAST include, e.g., well-tolerated,pharmaceutically acceptable esters such as alkyl esters includingmethyl, ethyl, propyl, isopropyl, butyl, isobutyl or pentyl esters.Additional esters such as phenyl-C₁-C₅ alkyl esters may be used,although methyl ester is preferred.

Methods for synthesizing prodrugs are described in the following reviewson the subject, which are incorporated herein by reference for theirdescription of these methods:

-   -   Higuchi, T.; Stella, V. eds. Prodrugs as Novel Drug Delivery        Systems. ACS Symposium Series. American Chemical Society:        Washington, D.C. (1975).    -   Roche, E. B. Design of Biopharmaceutical Properties through        Prodrugs and Analogs. American Pharmaceutical Association:        Washington, D.C. (1977).    -   Sinkula, A. A.; Yalkowsky, S. H. J Pharm Sci. 1975, 64, 181-210.    -   Stella, V. J.; Charman, W. N. Naringrekar, V. H. Drugs 1985, 29,        455-473.    -   Bundgaard, H., ed. Design of Prodrugs. Elsevier: New York        (1985).    -   Stella, V. J.; Himmelstein, K. J. J. Med. Chem. 1980, 23,        1275-1282.    -   Han, H-K; Amidon, G. L. AAPS Pharmsci 2000, 2, 1-11.    -   Denny, W. A. Eur. J. Med. Chem. 2001, 36, 577-595.    -   Wermuth, C. G. in Wermuth, C. G. ed. The Practice of Medicinal        Chemistry Academic Press: San Diego (1996), 697-715.    -   Balant, L. P.; Doelker, E. in Wolff, M. E. ed. Burgers Medicinal        Chemistry And Drug Discovery John Wiley & Sons: New York (1997),        949-982.

Formula I is as follows:

Examples of the preparation of DAST, salts thereof and pharmaceuticalcompositions thereof follow.

Preparation of the Intermediate: 4-amino-3-fluorophenol

To a dry flask purged with Argon was added 10% Pd/C (80 mg) followed by3-fluoro-4-nitrophenol (1.2 g, 7.64 mmol) as a solution in ethyl acetate(40 mL). The mixture was stirred under an H₂ atmosphere for 4 h. Themixture was filtered through a pad of Celite and the solvent wasevaporated under reduced pressure to afford the desired product as a tansolid (940 mg, 7.39 mmol; 97% yield); ¹H-NMR (DMSO-d₆) 4.38 (s, 2H),6.29-6.35 (m, 1 H), 6.41 (dd, J=2.5, 12.7, 1H), 6.52-6.62 (m, 1H), 8.76(s, 1H)

Preparation of the Starting Material 1:4-(4-amino-3-fluorophenoxy)pyridine-2-carboxylic Acid Methylamide

A solution of intermediate 4-amino-3-fluorophenol, (500 mg, 3.9 mmol) inN,N-dimethylacetamide (6 mL) cooled to 0° C. was treated with potassiumtert-butoxide (441 mg, 3.9 mmol), and the brown solution was allowed tostir at 0° C. for 25 min. To the mixture was added4-chloro-N-methyl-2-pyridinecarboxamide, (516 mg, 3.0 mmol) as asolution in dimethylacetamide (4 mL). The reaction was heated at 100° C.for 16 h. The mixture was cooled to room temperature, quenched with H₂O(20 mL), and extracted with ehtylacetate (4×40 mL). The combinedorganics were washed with H₂O (2×30 mL), dried (MgSO₄), and evaporatedto afford a red-brown oil. ¹H-NMR indicated the presence of residualdimethylacetamide, thus the oil was taken up in diethylether (50 mL) andwas further washed with brine (5×30 mL). The organic layer was dried(MgSO₄) and concentrated to give 950 mg of the desired product, startingmaterial 1, as a red-brown solid, which was used in the next stepwithout purification.

Example 1: Preparation of DAST:4{4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-fluorophenoxy}-pyridine-2-carboxylicAcid Methylamide

To a solution of 4-(4-amino-3-fluorophenoxy)pyridine-2-carboxylic acidmethylamide (starting material 1, 177 mg, 0.68 mmol) in toluene (3 mL)was added 4-chloro-3-(trifluoromethyl)phenyl isocyanate (150 mg, 0.68mmol). The mixture was stirred at room temperature for 72 h. Thereaction was concentrated under reduced pressure and the residue wastriturated with diethylether. The resulting solid was collected byfiltration and dried in vacuo for 4 h to afford the title compound (155mg, 0.32 mmol; 47% yield); ¹H-NMR (DMSO-d₆) 2.78 (d, J=4.9, 3H),7.03-7.08 (m, 1H), 7.16 (dd, J=2.6, 5.6, 1H), 7.32 (dd, J=2.7, 11.6,1H), 7.39 (d, J=2.5, 1H), 7.60 (s, 2H), 8.07-8.18 (m, 2H), 8.50 (d,J=5.7, 1H), 8.72 (s, 1H), 8.74-8.80 (m, 1H), 9.50 (s, 1H); MS (HPLC/ES)483.06 m/z =(M+1).

Example 2: Preparation of the Salt:4{4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-fluorophenoxy}-pyridine-2-carboxylicAcid Methylamide Hydrochloride

The compound of Example 1 as a free base (2.0 g) was dissolved inanhydrous tetrahydrofuran (15 mL) and a 4M HCl/dioxane was added(excess). The solution was then concentrated in vacuo to afford 2.32grams of off-white solids. The crude salt was dissolved in hot ethanol(125 mL), activated carbon was added and the mixture heated at refluxfor 15 minutes. The hot suspension was filtered through a pad of Celite521 and allowed to cool to room temperature. The flask was placed in afreezer overnight. The crystalline solids were collected by suctionfiltration, washed with ethanol, then hexane and air-dried. The motherliquors were concentrated down and crystallization (in freezer) allowedtaking place overnight. A second crop of solids was collected andcombined with the first crop. The colorless salt was dried in a vacuumoven at 60° C. over two days. Yield of hydrochloride salt obtained 1.72g (79%).

Melting point: 215° C.Elemental analysis:

Calcd. Found C 48.57 48.68 H 3.11 2.76 N 10.79 10.60 Cl 13.65 13.63 F14.63 14.88

Example 3: Preparation of the Salt:4{4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-fluorophenoxy}-pyridine-2-carboxylicAcid Methylamide Mesylate

The compound of Example 1 as a free base (2.25 g) was dissolved inethanol (100 mL) and a stock solution of methanesulfonic acid (excess)was added. The solution was then concentrated in vacuo to afford ayellow oil. Ethanol was added and concentration repeated, affording 2.41g of off-white solids. The crude salt was dissolved in hot ethanol (˜125mL) and then cooled slowly to crystallize. After reaching roomtemperature, the flask was placed in a freezer overnight. The colorlesscrystalline material was collected by suction filtration; the filtercake was washed with ethanol, then hexane and air-dried, to afford 2.05g of material, which was dried in a vacuum oven at 60° C. overnight.

Melting point: 231° C.Elemental analysis:

Calcd. Found C 45.64 45.34 H 3.31 3.08 N 9.68 9.44 Cl 6.12 6.08 F 13.1313.42 S 5.54 5.59

Example 4: Preparation of the Salt:4{4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-fluorophenoxy}-pyridine-2-carboxylicAcid Methylamide Phenylsulfonate

The compound of Example 1 as a free base (2.25 g) was suspended inethanol (50 mL) and benzensulfonic acid (0.737 g) in ethanol (50 mL) wasadded. The mixture was heated with vigorous stirring. All solid materialdissolved to give a reddish solution. The solution was allowed to coolto room temperature and the flask scratched. Crystal formation was slow,some seeds were found, added to solution and placed in freezerovernight. Grayish-tan solids had formed in the flask; the material wasbroken up & collected by suction filtration. The solids were washed withethanol, then hexane and air-dried. Weighed product: 2.05 g, 69% yield.

Melting point: 213° C.

Elemental Analysis:

Calcd. Found C 50.59 50.24 H 3.30 3.50 N 8.74 8.54 F 11.86 11.79 Cl 5.535.63 S 5.00 5.16

Example 5: Preparation of a 1+4 Solid Dispersion of4{4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-fluorophenoxy}-pyridine-2-carboxylicAcid Methyl Amide with Polyvinylpyrrolidone

In an uncapped vial, one part of the compound of Example 1 as a freebase was mixed with four parts polyvinylpyrrolidone (PVP-25/Kollidon®25), and dissolved in a sufficient amount of a 1:1 mixture of acetoneand ethanol, until all powders are in solution. The uncapped vial wasplaced into a vacuum oven set at 40° C., and let dry for at least 24-48hours.

Example 6: Preparation of a 1+3 Solid Dispersion of4{4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-fluorophenoxy}-pyridine-2-carboxylicAcid Methyl Amide with Polyvinylpyrrolidone

One part of the compound of Formula I as base and three parts ofpolyvinylpyrrolidone (PVP 25/Kollidon® 25) were dissolved in 30 parts ofa 80:20 acetone/ethanol mixture (w/w). Using a rotary vacuum evaporatorthe solvent was removed at 70° C. The dry residue was removed from theevaporation flask and sieved (630 μm).

Example 7: Preparation of a 1+7 Solid Dispersion of4{4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-fluorophenoxy}-pyridine-2-carboxylicAcid Methyl Amide with Polyvinylpyrrolidone

One part of the compound of Formula I as base and seven parts PVP 25were dissolved in 30 parts of a 80:20 acetone/ethanol mixture (w/w).Using a rotary vacuum evaporator the solvent was removed at 70° C. Thedry residue was removed from the evaporation flask and sieved (630 μm).

Example 8: Solid Dispersion of4{4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-fluorophenoxy}-pyridine-2-carboxylicAcid Methyl Amide with Hydroxypropyl Cellulose (HPC) Prepared by MeltExtrusion

Two parts of the compound of Formula I as base were mixed with one partof Maltitol and seven parts of HPC-M. The mixture was extruded using alab twin screw extruder at a temperature of 160-200° C. The extrudedmaterial was cut and subsequently milled using an impact lab mill. Theresulting powder can be used as it is or it can be further formulatedfor example to sachet, capsule or tablet formulations.

Example 9: Solid Dispersion of4{4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-fluorophenoxy}-pyridine-2-carboxylicAcid Methyl Amide with PVP and Croscarmellose Sodium

A solution of 0.4 kg of the of the compound of Formula I as base and 1.2kg of PVP 25 in a mixture of 6.4 kg acetone and 1.6 kg ethanol wasprepared. Using a fluidized bed vacuum granulator this solution wassprayed onto a powder bed of 1.6 kg croscarmellose sodium at atemperature of 60-70° C. After drying the product was sieved (1 mm). Thegranulate can be used as it is or it can be further formulated forexample to sachet, capsule or tablet formulations.

Example 10: Solid Dispersion of4{4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-fluorophenoxy}-pyridine-2-carboxylicAcid Methyl Amide with PVP and Sodium Starch Glycolate

This material was prepared in a similar way as described in Example 9,except that the solution is sprayed onto a powder bed of 1.6 kg sodiumstarch gycolate Type A (Explotab®)

Example 11: Solid Dispersion of4{4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-fluorophenoxy}-pyridine-2-carboxylicAcid Methyl Amide with PVP and Croscarmellose Sodium

A solution of 0.4 kg of the of the compound of Formula I as base and 1.6kg of PVP 25 in a mixture of 6.4 kg acetone and 1.6 kg ethanol wasprepared. Using a fluidized bed vacuum granulator this solution wassprayed onto a powder bed of 2 kg croscarmellose sodium at a temperatureof 60-70° C. After drying the product was sieved (1 mm). The granulatecan be used as it is or it can be further formulated for example tosachet, capsule or tablet formulations.

Example 12: Solid Dispersion of4{4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-fluorophenoxy}-pyridine-2-carboxylicAcid Methyl Amide with PVP, Croscarmellose Sodium and MicrocrystallineCellulose

This material was prepared in a similar way as described in Example 11,except that the solution was sprayed onto a powder bed consisting of 1kg croscarmellose sodium and 1 kg microcrystalline cellulose.

Example 13: Solid Dispersion of4{4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-fluorophenoxy}-pyridine-2-carboxylicAcid Methyl Amide with HPC-SL and Croscarmellose Sodium

A solution of 0.4 kg of the of the compound of Formula I as base and 1.6kg of HPC-SL in 20 kg acetone was prepared. Using a fluidized bed vacuumgranulator this solution was sprayed onto a powder bed of 2 kgcroscarmellose sodium at a temperature of 40-60° C. After drying theproduct was sieved (1 mm). The granulate can be used as it is or it canbe further formulated for example to sachet, capsule or tabletformulations.

Example 14: Solid Dispersion of4{4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-fluorophenoxy}-pyridine-2-carboxylicAcid Methyl Amide with HPC-L and Croscarmellose Sodium

A solution of 0.4 kg of the of the compound of Formula I as base and 1.6kg of HPC-L in 28 kg acetone was prepared. Using a fluidized bed vacuumgranulator this solution was sprayed onto a powder bed of 2 kgcroscarmellose sodium at a temperature of 40-60° C. After drying theproduct was sieved (1 mm). The granulate can be used as it is or it canbe further formulated for example to sachet, capsule or tabletformulations.

Example 15: Tablets Containing a Solid Dispersion of4{4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-fluorophenoxy}-pyridine-2-carboxylicAcid Methyl Amide

The granulate of Example 11 was roller compacted and screened 3 and 1mm. Subsequently the compacted granulate was blended with 0.54 kgcroscarmellose sodium, 24 g colloidal anhydrous silica and 36 gmagnesium stearate. This ready-to-press blend was compressed on a rotarytablet press to tablets containing 20, 50 an 100 mg of the compound ofFormula I. The tablets may be film-coated for light protection.

Example 16: Tablets Containing a Solid Dispersion of4{4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-fluorophenoxy}-pyridine-2-carboxylicAcid Methyl Amide

The granulate of Example 12 was roller compacted and screened 3 and 1mm. Subsequently the compacted granulate was blended with 0.54 kgcroscarmellose sodium, 24 g colloidal anhydrous silica and 36 gmagnesium stearate. This ready-to-press blend was compressed on a rotarytablet press to tablets containing 20, 50 an 100 mg of the compound ofFormula I. The tablets may be film-coated for light protection.

Example 17: Tablets Containing a Solid Dispersion of4{4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-fluorophenoxy}-pyridine-2-carboxylicAcid Methyl Amide

A solution of 0.4 kg of the of the compound of Formula I as base and 1.2kg of PVP 25 in a mixture of 6.4 kg acetone and 1.6 kg ethanol wasprepared. Using a fluidized bed vacuum granulator this solution wassprayed onto a powder bed consisting of 0.8 kg croscarmellose sodium and0.8 kg microcrystalline cellulose at a temperature of 60-70° C. Afterdrying the product is sieved (1 mm). The granulate is roller compactedand screened 3 and 1 mm. Subsequently the compacted granulate wasblended with 1.34 kg croscarmellose sodium, 24 g colloidal anhydroussilica and 36 g magnesium stearate. This ready-to-press blend iscompressed on a rotary tablet press to tablets containing 20, 50 an 100mg of the compound of Formula I. The tablets may be film-coated forlight protection.

The specific dose level and frequency of dosage may vary, depending upona variety of factors, including the activity of the active agent, itsmetabolic stability and length of action, rate of excretion, mode andtime of administration, the age, body weight, health condition, gender,diet, baseline hematologic and biologic parameters (e.g., WBCs,granulocytes, platelets, hemoglobin, creatinine, bilirubin, albumin,etc.), etc., of the subject, and the severity, intensity, stage of thecancer, primary site of cancer, size of cancer lesion, presence orextent of metastases, surgical status, disease progression (i.e.,aggressive), etc. of the disease.

DAST can be administered in any form by any effective route, including,e.g., oral, parenteral, enteral, intraperitoneal, topical, transdermal(e.g., using any standard patch), ophthalmic, nasally, local, non-oral,such as aerosol, spray, inhalation, subcutaneous, intravenous,intramuscular, buccal, sublingual, rectal, vaginal, intra-arterial,intrathecal, intratumoral, etc. DAST can be administered directly to thesite of a tumor, either pre- or post-operatively. It can be administeredalone, or in combination with any ingredient(s), active or inactive.

DAST can be administered by the oral route using the pharmaceuticalcomposition of the present invention. Dosages will generally range,based on body weight, from about 0.01 mg/kg to about 50 mg/kg; fromabout 1 mg/kg to about 40 mg/kg; from about 5 mg/kg to about 30 mg/kg;from about 10 to about 25 mg/kg; about 10 mg/kg; about 20 mg/kg; about25 mg/kg; about 30 mg/kg; etc.

Any suitable dosing interval can be used in accordance with the presentinvention. For example, DAST can be administered once, twice (BID),three, four, etc., times a day. For example, about 100, about 200, about400 mg, about 500 mg, about 600 mg, or about 800 mg can be administeredone, twice, or three times daily.

DAST can be administered at any suitable time. For example, it can beadministered routinely as other chemotherapeutic agents; it can beadministered as a bolus prior to a surgical intervention; prior to orafter radiation, radiofrequency ablation and other energy treatments;post-operatively; pre-operatively; etc.

DAST can be further combined with any other suitable additive orpharmaceutically acceptable carrier. Such additives include any of thoseused conventionally, such as those described in Remington: The Scienceand Practice of Pharmacy (Gennaro and Gennaro, eds, 20th edition,Lippincott Williams & Wilkins, 2000); Theory and Practice of IndustrialPharmacy (Lachman et al., eds., 3rd edition, Lippincott Williams &Wilkins, 1986); Encyclopedia of Pharmaceutical Technology (Swarbrick andBoylan, eds., 2nd edition, Marcel Dekker, 2002).

DAST and the pharmaceutical compositions of the present invention can bein any suitable form, without limitation. Forms suitable for oral use,include, but are not limited to, tablets, troches, lozenges, aqueous oroily suspensions, dispersible powders or granules, emulsions, hard orsoft capsules, solutions, syrups and elixirs. Compositions intended fororal use may be prepared according to any method known to the art forthe manufacture of pharmaceutical compositions.

DAST can be formulated with other ingredients, e.g., “pharmaceuticallyacceptable carriers” or “excipients” to indicate they are combined withthe active drug and can be administered safely to a subject fortherapeutic purposes. These include, but are not limited to,antioxidants, preservatives, dyes, tablet-coating compositions,plasticizers, inert carriers, excipients, polymers, coating materials,osmotic barriers, devices and agents which slow or retard solubility,etc.

Pharmaceutical compositions intended for oral use may be preparedaccording to any suitable method known to the art for the manufacture ofpharmaceutical compositions. Such compositions may contain one or moreagents selected from the group consisting of diluents, sweeteningagents, flavoring agents, coloring agents and preserving agents in orderto provide palatable preparations.

Non-toxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients may be, for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; and binding agents, forexample magnesium stearate, stearic acid or talc.

Pharmaceutical compositions for oral use may also be presented as hardgelatin capsules wherein the active ingredient is mixed with an inertsolid diluent, for example, calcium carbonate, calcium phosphate orkaolin, or as soft gelatin capsules wherein the active ingredient ismixed with water or an oil medium, for example peanut oil, liquidparaffin or olive oil.

Aqueous suspensions containing the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions may alsobe used. Such excipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethylene oxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolsuch as polyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides, for example polyethylene sorbitan monooleate. The aqueoussuspensions may also contain one or more preservatives, for exampleethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, oneor more flavoring agents, and one or more sweetening agents, such assucrose or saccharin.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example, sweetening, flavoring and coloringagents, may also be present.

DAST and the pharmaceutical compositions of the present invention mayalso be in the form of non-aqueous liquid formulations, e.g., oilysuspensions which may be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orpeanut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide palatable oralpreparations. These compositions may be preserved by the addition of ananti-oxidant such as ascorbic acid.

DAST and the pharmaceutical compositions of the invention may also be inthe form of oil-in-water emulsions. The oily phase may be a vegetableoil, for example olive oil or arachis oil, or a mineral oil, for exampleliquid paraffin or mixtures of these. Suitable emulsifying agents may benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitolanhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, a preservative and flavoring and coloringagents.

DAST and the pharmaceutical compositions of the invention may also beadministered in the form of suppositories for rectal or vaginaladministration of the drug. These compositions can be prepared by mixingthe drug with a suitable non-irritating excipient which is solid atordinary temperatures but liquid at the rectal temperature or vaginaltemperature and will therefore melt in the rectum or vagina to releasethe drug. Such materials include cocoa butter and polyethylene glycols.

DAST and the pharmaceutical compositions of the invention may also beadministrated transdermally using methods known to those skilled in theart (see, for example: Chien; “Transdermal Controlled SystemicMedications”; Marcel Dekker, Inc.; 1987. Lipp et al. WO94/04157). Forexample, a solution or suspension of a compound of Formula I in asuitable volatile solvent optionally containing penetration enhancingagents can be combined with additional additives known to those skilledin the art, such as matrix materials and bacteriocides. Aftersterilization, the resulting mixture can be formulated following knownprocedures into dosage forms. In addition, on treatment with emulsifyingagents and water, a solution or suspension of a compound of Formula Imay be formulated into a lotion or salve.

Suitable solvents for processing transdermal delivery systems are knownto those skilled in the art, and include lower alcohols such as ethanolor isopropyl alcohol, lower ketones such as acetone, lower carboxylicacid esters such as ethyl acetate, polar ethers such as tetrahydrofuran,lower hydrocarbons such as hexane, cyclohexane or benzene, orhalogenated hydrocarbons such as dichloromethane, chloroform,trichlorotrifluoroethane, or trichlorofluoroethane. Suitable solventsmay also include mixtures of one or more materials selected from loweralcohols, lower ketones, lower carboxylic acid esters, polar ethers,lower hydrocarbons, halogenated hydrocarbons.

Suitable penetration enhancing materials for transdermal delivery systemare known to those skilled in the art, and include, for example,monohydroxy or polyhydroxy alcohols such as ethanol, propylene glycol orbenzyl alcohol, saturated or unsaturated C8-C18 fatty alcohols such aslauryl alcohol or cetyl alcohol, saturated or unsaturated C8-C18 fattyacids such as stearic acid, saturated or unsaturated fatty esters withup to 24 carbons such as methyl, ethyl, propyl, isopropyl, n-butyl,sec-butyl, isobutyl, tertbutyl or monoglycerin esters of acetic acid,capronic acid, lauric acid, myristinic acid, stearic acid, or palmiticacid, or diesters of saturated or unsaturated dicarboxylic acids with atotal of up to 24 carbons such as diisopropyl adipate, diisobutyladipate, diisopropyl sebacate, diisopropyl maleate, or diisopropylfumarate. Additional penetration enhancing materials includephosphatidyl derivatives such as lecithin or cephalin, terpenes, amides,ketones, ureas and their derivatives, and ethers such as dimethylisosorbid and diethyleneglycol monoethyl ether. Suitable penetrationenhancing formulations may also include mixtures of one or morematerials selected from monohydroxy or polyhydroxy alcohols, saturatedor unsaturated C8-C18 fatty alcohols, saturated or unsaturated C8-C18fatty acids, saturated or unsaturated fatty esters with up to 24carbons, diesters of saturated or unsaturated discarboxylic acids with atotal of up to 24 carbons, phosphatidyl derivatives, terpenes, amides,ketones, ureas and their derivatives, and ethers.

Suitable binding materials for transdermal delivery systems are known tothose skilled in the art and include polyacrylates, silicones,polyurethanes, block polymers, styrenebutadiene copolymers, and naturaland synthetic rubbers. Cellulose ethers, derivatized polyethylenes, andsilicates may also be used as matrix components. Additional additives,such as viscous resins or oils may be added to increase the viscosity ofthe matrix.

Compositions comprising precursors can also be formulated for controlledrelease, where release of the active ingredient is regulated ormodulated to achieve a desired rate of delivery into the systemiccirculation. A controlled release formulation can be pulsed, delayed,extended, slow, steady, immediate, rapid, fast, etc. It can comprise oneor more release formulations, e.g. extended- and immediate-releasecomponents. Extended delivery systems can be utilized to achieve adosing internal of once every 24 hours, once every 12 hours, once every8 hours, once every 6 hours, etc. The dosage form/delivery system can bea tablet or a capsule suited for extended release, but a sustainedrelease liquid or suspension can also be used. A controlled releasepharmaceutical formulation can be produced which maintains the releaseof, and or peak blood plasma levels of DAST.

In preferred solid oral pharmaceutical compositions according to theinvention, at least 25% of DAST exists as a coprecipitate, morepreferable at least 40% of DAST exists as a coprecipitate.

Micronization can be achieved by standard milling methods, preferably byair chat milling, known to a skilled person. The micronized form canhave a mean particle size of from 0.5 to 10 μm, preferably from 1 to 6μm, more preferably from 1 to 3 μm. The indicated particle size is themean of the particle size distribution measured by laser diffractionknown to a skilled person (measuring device: HELOS, Sympatec).

Pharmaceutical compositions which are preferred comprise DAST in aportion of at least 25%, preferably at least 45%, more preferably atleast 50%, even more preferably at least 55%, by weight of thecomposition. Amounts of at least 62%, or at least 69%, or at least 75%by weight of the composition can be used under certain circumstances.Methods for preparing such formulations are disclosed in publishedinternational applications WO05/009961, published Feb. 3, 2005, andWO06/026500, published March 9, 2006, which are incorporated herein byreference.

Without further elaboration, it is believed that one skilled in the art,using the preceding description, can utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever. The entiredisclosure of all applications, patents and publications, cited aboveand in the figures are hereby incorporated by reference in theirentirety.

Biological Examples

Generation of c-KIT-Expressing Ba/F3 Cell Lines

The cDNA encoding full length human c-KIT with a deletion in exon 11that removed amino acid residues 557-558 was ligated into the mammalianexpression vector pClneo (Promega). Imatinib (Gleevec) -resistantvariants of the KIT exon 11 deletion mutant were generated. Allmutations were confirmed by DNA sequencing.

The expression vectors encoding the c-KIT exon 11 mutant or its Imatinib(Gleevec) -resistant mutant variants were transfected into Ba/F3 cellsby electroporation. Selective pressure was applied to the transfectedcells by removing IL-3 from the culture medium. After IL-independentpopulations, further selective pressure was applied by also growing thecells in the presence of 1 mg/mL G418. The resulting stable pools ofBa/F3 cells were found to express c-KIT by western blot using anantibody specific for c-KIT. The stable pools were further characterizedby sequencing genomic DNA to confirm the presence of the transfectedc-KIT cDNA.

Cell Proliferation Assay

This assay utilizes cellular ATP as a marker for cellproliferation/viability. On day 1, Ba/F3 cells were plated in 96 welldishes (Costar 3603) at 10,000 cells per well in 10% FBS in RPMI mediumwith 1 mg/ml G418. Test compounds, serially diluted in the same mediumat 10' for an eight-point dose response to give rise to finalconcentrations ranging from 0.6 to 10,000 nM, were added to the cells.Plates were then incubated in a 5% CO2 incubator at 37° C. for 3 days.After 72 h, 100 microliters of lysis/luciferase reagent (CellTiter-Glo,Promega G7573) was added to each well. The cells were then incubated ona shaker for 5 minutes at room temperature, and luminescence wasmeasured on a Victor 5 (Perkin Elmer) spectrophotometer.

Growth inhibition was measured by comparing luminescence signal fromtreated vs. untreated cells in assay plates, and the IC50 analysis ofcell proliferation inhibition by compounds was analyzed using Analyze 5in-house software. IC50 values obtained for Imatinib (Gleevec) andNexavar in the various c-KIT-expressing Ba/F3cell lines are summarizedin Table 1. The IC50 values are mean values calculated from at leastthree experiments.

TABLE 1 Average IC₅₀ [nM] for c-KIT-expressing Ba/F3 cellularproliferation, n ≥ 3 Exon Eson Exon Exon Exon Exon 11 11 + 11 11 + 11 +11 + Inhibitor Deletion V654A +T6701 D816G N822K Y823D Imatinib 5168 >10,000 87 221 295 (Gleevec) DAST 7 38 19 14 12  129*

TABLE 2 K  P  M  Y  E  V  Q  W  K  V  V  E  E  I  N  G50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65N  N  Y  V  Y  I  D  P  T  Q  L  P  Y  D  H66 67 68 69 70 71 72 73 74 75 76 77 78 79 80

1-29. (canceled)
 30. A method to determine the ability of DAST to treata hyper-proliferative disease, such as cancer, with acquired resistanceto a KIT inhibitor, comprising culturing cancer cells in the presence ofDAST, wherein DAST is4{4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-fluorophenoxy}-pyridine-2-carboxylicacid methylamide or a polymorph, hydrate, pharmaceutically acceptablesalt, metabolite, prodrug or solvate thereof, or a combination thereof.31. A method according to claim 30, wherein the cells express adouble-mutation.
 32. A method according to claim 30, wherein the cellsexpress the constitutively active KIT polypeptide.
 33. A method tosuggest cancer treatment for a patient, comprising taking a cancer cellsample from said patient, test the cells in the method according toclaim 30, and if cells die as a result of the test, determine that thecells are sensitive to DAST and thus useful in treating the patient whohas acquired resistance to the KIT inhibitor.
 34. A method ofdetermining whether to treat a subject having cancer with DAST,comprising determining the presence of a mutation in a KIT gene, whereinsaid mutation is an activating and/or KIT-inhibitor resistance mutation,wherein DAST is4{4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-fluorophenoxy}-pyridine-2-carboxylicacid methylamide or a polymorph, hydrate, pharmaceutically acceptablesalt, metabolite, prodrug or solvate thereof, or a combination thereof.35. A method according to claim 34, further comprising suggesting theadministration of DAST to the subject.
 36. A method according to claim34, further comprising the administration of DAST to the subject.
 37. Amethod for enhancing a patient's quality of life who has cancer, whichpatient has a secondary mutation that is one or more of i) deletion ofamino acid residues 557-558; ii) deletion of amino acid residues551-555; iii) deletion of amino acid residues 550-558; iv) deletion ofamino acid residues 559-560; v) deletion of amino acid residues 557-561;vi) deletion of amino acid residues 554-558; vii) deletion of amino acidresidues 552-557; viii) mutations at residue 559, including V559D,V559A, or V559G; ix) mutations at residue 560, including V560D, V560E,or V560G; x) W557S, alone, or in combination with a deletion of aminoacids 552-556; xi) mutations at amino acid residue 557, including W557R;and xii) mutations at amino acid residue 576, including L576P,comprising administering to said patient an effective amount of DAST,wherein DAST is4{4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-fluorophenoxy}-pyridine-2-carboxylicacid methylamide or a polymorph, hydrate, pharmaceutically acceptablesalt, metabolite, prodrug or solvate thereof, or a combination thereof.38. A method according to claim 37, wherein the DAST is in the form of asolid dispersion.
 39. A method according to claim 37, wherein the DASTis administered with sugar.
 40. A method according to claim 37, whereinthe DAST is in a preparation that is colored to provide a palatablepreparation.
 41. A method according to claim 37, wherein the cancer isin the form of an unspecified adult solid tumor.
 42. A method accordingto claim 37, wherein the DAST is in a preparation that has beenformulated with ethanol and is given orally.
 43. A method according toclaim 37, wherein to reduce undesirable side effects, the DAST has beenmodified with one or more labile functional groups.
 44. A methodaccording to claim 37, wherein the DAST has been formulated forcontrolled release, where release of the active ingredient is regulatedor modulated to achieve a desired rate of delivery into the systemiccirculation, preferably rapid or fast release.
 45. Use of DAST accordingto claim 37, wherein the cancer was initially sensitive to a derivativeof imatinib mesylate and acquired resistance to a derivative of imatinibmesylate, other than a salt of imatinib mesylate, and wherein aneffective amount of DAST is administered to the patient.
 46. Useaccording to claim 45, further comprising additionally administeringimatinib mesylate, a derivative of imatinib mesylate, or a salt ofimatinib mesylate.
 47. DAST, which is used according to claim
 37. 48.DAST, which is used together with imatinib mesylate, a derivative ofimatinib mesylate, or a salt of imatinib mesylate as recited in claim45.